Inactivated avian polyomavirus vaccine in psittacine birds

ABSTRACT

The present invention provides a vaccine which is protective against avian polyomavirus infection in a bird which is classified as being a member of the Psittaciformes order which comprises an immunogenic amount of an inactivated avian polyomavirus in a pharmaceutically acceptable carrier. Also provided is an adjuvant suitable for use in a bird which is classified as being a member of the Psittaciformes order. Further provided is a composition which produces an anamnestic response against avian polyomavirus infection in a sensitized bird which is classified as being a member of the Psittaciformes order, which comprises an anamnestic response inducing amount of a recombinant protein (e.g., VP1 capsid protein) of avian polyomavirus in a pharmaceutically acceptable carrier. Methods are also provided for preventing avian polyomavirus infection in a bird, or in several different species of birds, classified as being a member of the Psittaciformes order, comprising administering a vaccine comprising an immunogenic amount of an inactivated avian polyomavirus and a pharmaceutically acceptable carrier.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an avian polyomavirus vaccine and to amethod of preventing avian polyomavirus infection in Psittaciformes.

2. Background Art

The first acute, generalized infection associated with avianpolyomavirus was described in 1980 in young psittacine birds and wascalled budgerigar fledgling disease (Davis, R. B., et al., "A viraldisease of fledgling budgerigars," Avian Dis., 1981, 25:179-183;Bozeman, L. H., et al., "Characterization of a papovavirus isolated fromfledgling budgerigars," Avian Dis., 1981, 25:972-980; Bernier, G., etal., "A generalized inclusion body disease in the budgerigar(Melopsittacus undulatus) caused by a papovavirus-like agent," AvianDis., 1981, 25:1083-1092; Dykstra, M. J., et al., "Investigations ofbudgerigar fledgling disease virus," Am. J. Vet. Res., 1984,45:1883-1887; Lehn, H., Muller, H., "Cloning and characterization ofbudgerigar fledgling disease virus (BFDV), an avian polyomavirus,"Virology, 1986, 151:362-370). Since its discovery in 1980, avianpolyomavirus has been associated with disease in a number of differentspecies of companion and aviary birds including Budgerigars, caiques,macaws, Amazon parrots, conures, cockatoos, lovebirds, SplendidParakeets, Pionus Parrots, African Grey Parrots, Eclectus Parrots,Cockatiels, finches and lories (Davis et al., 1981; Bozeman et al.,1981; Bernier et al., 1981; Lehn and Muller, 1986; Jacobson, E. R., etal., "Epornitic of papova-like virus-associated disease in a psittacinenursery," J. Am. Vet. Med. Assoc., 1984, 185:1337-1341; Clubb, S. L.,Davis, R. B., "Outbreak of papova-like viral infection in a psittacinenursery-a retrospective view," Proc. Assoc. Avian Vet., Toronto, 1984,121-129; Graham, D. L., "An update on selected pet bird virusinfections," Proc. Assoc. Avian Vet., Toronto, 1984, 267-280; Gaskin, J.M., "Psittacine viral disease: A perspective," J. Zoo. Wildl. Med.,1989, 20:249-264; Johnston, K. M., Riddell, C., "Intranuclear inclusionbodies in finches," Can. Vet. J., 1986, 27:432-434; Marshall, R.,"Papova-like virus in a finch aviary," Proc. Assoc. Avian Vet., 1989,203-207; Schmidt, R. E., et al., "Morphologic identification ofpapovavirus in a Moluccan cockatoo (Cacatua moluceensis) with neurologicsigns," Assoc. Avian Vet. Today, 1987, 1:107-108; Pass, D. A., et al.,"A papova-like virus infection of splendid parakeets (Neophemasplendida)," Avian Dis., 1987, 31:680-684; Pass, D. A., "A papova-likevirus infection of lovebirds (Agapornis sp.)," Aust. Vet. J.; 1985,82:318-319).

The type of clinical disease in Budgerigars, for example, depends uponthe age and condition of the bird when exposure to the virus occurs.Neonates from infected flocks may develop normally for 10-15 days andthen suddenly die with no premonitory signs. Other infected hatchlingsmay develop clinical signs that include abdominal distention,subcutaneous hemorrhage, tremors of the head and neck, ataxia andreduced formation of down and contour feathers (Hirai, K., et al.,"Isolation of a papova-like agent from young budgerigars with featherabnormalities," J. Vet. Sci. 46:577-587, 1984; Bernier et al., 1984;Clubb and Davis, 1984; Schmidt et al., 1987; Histopathology Reports#SC90-0637 and #SC90-0638, Schubot Exotic Bird Health Center, Texas A&MUniversity; Vernot, J., personal communication; Dykstra, M. J., Bozeman,L. H., "A light and electron microscopic examination of budgerigarfledgling disease virus in tissue and in cell culture, Avian Pathol.11:11-18, 1982). Infections have also been associated with decreasedhatchability and embryonic death (Hudson, L., Hay, F. C., "Isolation andstructure of immunoglobulins," Hudson, L., Hay, F. C. Ed., PracticalImmunology, Boston, 1980, 156-202). Mortality rates can be as high as100% in affected hatchlings. Surviving birds often exhibit dystrophicprimary tail feathers, lack of down feathers on the back and abdomen,and lack of filoplumes on the head and neck. Additionally, survivingbirds with primary feather abnormalities are usually unable to fly.

In larger psittacine birds, polyomavirus infections may cause peracutedeath with no premonitory signs, or acute death after development ofclinical changes including depression, anorexia, weight loss, delayedcrop-emptying, regurgitation, diarrhea, dehydration, subcutaneoushemorrhages, dyspnea, polyuria, and posterior paresis and paralysis(Pass et al., 1987; Johnston and Riddell, 1986; Mathey, W. J., Cho, B.R., "Tremors of nestling budgerigars with budgerigar fledgling disease,"Proc. 33rd West. Poult. Dis. Conf., 1984, 102; Woods, L., "Papova-likevirus in a purple finch," J. Zoo. Wild. Med., 1989, 218-219; Gaskin, J.M., "The serodiagnosis of psittacine viral infections," Assoc. AvianVet., Honolulu, 1988, 7-10). Characteristic lesions associated with apolyomavirus infection have been demonstrated in companion birds fromthe United States (Jacobson et al., 1984; Clubb and Davis, 1984; Graham,1984), Canada (Gough, J. F., "Outbreaks of budgerigar fledgling diseasein three aviaries in Ontario," Can. Vet. J., 1989, 30:672-674, Bernieret al., 1984), Japan (Hirai et al., 1984), Italy (Pascucci, S., et al.,"Malattia da virus papova-simile nel papagallino ondulato (Melopsittacusundulatus), Clin. Med. (Milan), 1983, 106:38-41), Hungary (Sztojkov, V.,et al., "A hullamous papagaj (Melopsittacus Undulatus) papovavirusokozta megbetegedesenek hazai megallapitasa, Magy Allatorv Lapja 1985,40:59-63), Germany (Krautwald, M-E, Kaleta, E. F., "Relationship ofFrench moult and early virus induced mortality in nestling budgerigars,"Proc. 8th Intl. Cong. World Vet. Poult. Assoc., 1985, 115) and Australia(Pass et al., 1987; Pass, 1985).

Immunodiffusion and virus neutralization techniques have been used todemonstrate anti-polyomavirus antibodies in psittacine birds (Jacobsonet al., 1984; Clubb and Davis, 1984; Gaskin, 1989; Davis et al., 1981;Gaskin, 1988; Lynch, J., et al., "Isolation and experimentalchicken-embryo-inoculation studies with budgerigar papovavirus," AvianDis. 1984, 28:1135-1139; Wainwright, P. O., et al., "Serologicalevaluation of some psittaciformes for budgerigar fledgling diseasevirus," Avian Dis. 1987, 31:673-676). During epornitics in mixedpsittacine bird collections, infected survivors and asymptomatic birdsexposed to them have been shown to develop anti-polyomavirusneutralizing antibodies (Jacobson et al., 1984; Clubb and Davis, 1984;Wainwright et al., 1987). Seronegative young adult birds willseroconvert when housed adjacent to seropositive breeding adults,indicating that an antibody response does occur following naturalexposure to the virus (Jacobson et al., 1984; Clubb and Davis, 1984;Wainwright et al., 1987; Davis, R. B., "Budgerigar fledgling disease(BFD), 32nd West Poult. Dis. Conf., 1983, 104). However, prior to thepresent invention it had not been determined whether this antibodyresponse could be induced through vaccination or whether the resultingimmunologic response would be protective.

In the past, attempts at producing a vaccine against avian polyomavirushave been unsuccessful. The existence of subclinical infections andchronically infected carrier birds, coupled with a lack of understandingof the epidemiologic and pathophysiologic characteristics of infectionhave all contributed to the lack of success.

Consequently, avian polyomavirus infections continue to cause highlevels of mortality in companion and aviary birds, resulting inpsychological distress for clients and financial burdens foraviculturists and retail distributors despite discovery of the virusover 14 years ago. Therefore, there exists a long-felt need in the artfor a safe and effective vaccine against avian polyomavirus which iscross-protective against the disease in multiple species ofPsittaciformes.

Another problem associated with vaccine failure in Psittaciformes hasbeen the lack of a suitable adjuvant. Two killed oil-adjuvantedherpesvirus (Pacheco's disease virus) vaccines that were conditionallylicensed for use in Psittaciformes were found to cause unacceptablereactions in a number of vaccinates, particularly cockatoos (Davis etal., 1981; Bozeman et al., 1981; Bernier et al., 1981; Dykstra et al.,1984). These reactions were characterized by the formation of abscesses(subcutaneous inoculation) or muscle necrosis (IM inoculation). In somePsittaciformes, granulomatous or necrotizing lesions were not noteduntil several months after vaccination. In other birds, lesions werenoted within several weeks of administration of a booster vaccination(Davis et al., 1981; Bozeman et al., 1981; Schmidt et al., 1987). Insome cases, deaths have been associated with the use of oil-adjuvantedvaccines.

In general, the advantage of oil-adjuvanted vaccines in comparison toother immunization products is their ability to induce durable immunitywhen mixed with an inactivated antigen. However, the occurrence ofadverse reactions in some Psittaciformes vaccinated with oil-adjuvantedvaccines created the need for an alternative adjuvant for use withinactivated antigens intended for administration in this order of birds(Davis et al., 1981; Bozeman et al., 1981; Schmidt et al., 1987).Therefore, there exists a need in the art for a suitable adjuvant foruse in Psittaciformes which augments the immune response yet does notproduce an adverse reaction in the vaccinate.

SUMMARY OF THE INVENTION

The present invention satisfies the long-felt need in the art for a safeand effective vaccine to protect psittacine birds against avianpolyomavirus disease by providing a vaccine which is protective againstavian polyomavirus infection in a bird which is classified as being amember of the Psittaciformes order, comprising an immunogenic amount ofan inactivated avian polyomavirus and a pharmaceutically acceptablecarrier.

In one embodiment, the vaccine is derived from a strain of inactivatedavian polyomavirus known as the L4 strain. In another embodiment, theinvention provides a vaccine wherein immunogenic amount of theinactivated avian polyomavirus corresponds to a titer of between 10⁴.5TCID₅₀ and 10⁷ TCID₅₀ for the avian polyomavirus before inactivation,but especially about 10⁵.8 TCID₅₀.

The present invention also satisfies the need for a suitable adjuvantfor use in psittacine species by providing an adjuvant, e.g., a longchain polydispersed β (1,4) linked mannan polymer interspersed withO-acetylated groups such as ACEMANNAN, for use, not only in the vaccinesof the present invention, but also in other psittacine vaccines.

Also provided is a composition which produces an anamnestic responseagainst avian polyomavirus infection in a sensitized bird which isclassified as being a member of the Psittaciformes order, comprising ananamnestic response inducing amount of a recombinant protein of avianpolyomavirus and a pharmaceutically acceptable carrier. In oneembodiment, the composition comprises a recombinant VP1 capsid proteinof avian polyomavirus.

The present invention also provides a method of preventing avianpolyomavirus infection in a bird which is classified as being a memberof the Psittaciformes order, comprising administering to the bird avaccine comprising an immunogenic amount of an inactivated avianpolyomavirus and a pharmaceutically acceptable carrier. In oneembodiment, the method further comprises administering at least onebooster vaccine to the bird.

Further, the invention provides a method of preventing avianpolyomavirus infection in a bird from a species which is classified asbeing a member of the Psittaciformes order, comprising administering tothe bird a vaccine comprising an immunogenic amount of an inactivatedavian polyomavirus which infects a bird from a different species of thePsittaciformes order and a pharmaceutically acceptable carrier. Thevaccines and compositions provided by the invention can be utilized inthe methods provided herein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is more particularly described in the followingexamples which are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art.

The present invention provides a vaccine which is protective againstavian polyomavirus infection in a bird which is classified as being amember of the Psittaciformes order, comprising an immunogenic amount ofan inactivated avian polyomavirus and a pharmaceutically acceptablecarrier. The term "immunogenic amount" means an amount of an immunogen,i.e., the inactivated avian polyomavirus or a portion thereof, which issufficient to induce an immune response in the vaccinated bird and whichprotects the bird against active infection with avian polyomavirus uponexposure thereto.

The birds which can be treated by the invention can be any of thevarious species of birds which are classified as being members of thePsittaciformes order. Examples of such birds include, but are notlimited to, Budgerigars (Melopsittacus undulatus), caiques (e.g.,Pionites leucogaster leucogaster), macaws (e.g., Ara ararauna), Amazonparrots (e.g., Amazona ochrocephala auropalliata, conures (e.g.,Pyrrhara picta, Aratinga wagleri wagleri, Aratinga solstitialis,Aratinga guarouba, Aratinga holochlora rubritorquis or Aratingaacuticaudata acuticaudata), cockatoos (e.g., Cacatua moluccensis,Cacatua ducorps, Cacatua sulphura, Cacatua goffini or Cacatua alba),Splendid Parakeets (Neophema splendida), Pionus Parrots (Pionusmaximillani), African Grey Parrots (Psittacus erithacus erithacus,Eclectus Parrots (Electus roratus), Cockatiels (Nymphicus hollandicus)and parakeets (e.g. Psittacula krameri krameri). Specificallyexemplified by the invention is a vaccine which is protective againstavian polyomavirus infection in a bird which is classified as being amember of the Psittaciformes order, comprising an immunogenic amount ofan inactivated avian polyomavirus and a pharmaceutically acceptablecarrier wherein the bird is selected from the group consisting of amacaw, an Amazon parrot, a conure, a cockatoo, a Pionus Parrot, and anAfrican Grey Parrot.

Given the surprising fact provided by the invention that avianpolyomavirus can be prevented in multiple species of Psittaciformesutilizing a single strain of avian polyomavirus, it is contemplated thatthe vaccines of the present invention can be constructed from anyisolated strain of avian polyomavirus which infects a member of thePsittaciformes order by utilizing the methods taught herein. Forexample, the subject avian polyomavirus can be isolated and culturedutilizing the method taught by Bozeman et al., (1981) or by othermethods known in the art. Once isolated, the virus can be purified ifdesired, inactivated, the vaccine prepared and the immunogenic doseoptimized by the methods taught herein.

In one embodiment of the invention, the inactivated avian polyomavirusvaccine is derived from an isolated avian polyomavirus designated the"L4" strain. The L4 strain was isolated from an infected Budgerigar atthe University of Georgia College of Veterinary Medicine in 1981utilizing the method of Bozeman et al., 1981, and can be obtained fromthe Laboratory of Dr. Phil D. Lukert, College of Veterinary Medicine,University of Georgia, Athens, Ga. 30602.

One embodiment of the invention provides a vaccine which is protectiveagainst avian polyomavirus infection in a bird which is classified asbeing a member of the Psittaciformes order, comprising an immunogenicamount of an inactivated avian polyomavirus and a pharmaceuticallyacceptable carrier, wherein the immunogenic amount of the inactivatedavian polyomavirus corresponds to a tiler of between 10⁴.5 TCID₅₀ and10⁷ TCID₅₀ for the avian polyomavirus before inactivation.

In a presently preferred embodiment, the immunogenic amount of theinactivated avian polyomavirus corresponds to a titer of 10⁵.8 TCID₅₀for the avian polyomavirus before inactivation. As used herein, theimmunogenic amount is expressed in terms of "TCID₅₀ " titer which isgiven its common meaning in the art of a tissue culture infection dosewhich infects 50% of the cells of a tissue culture inoculum. Thus, theimmunogenic amount of any particular strain of inactivated avianpolyomavirus that is utilized to prepare the vaccines of the inventionis based upon the tissue culture infectivity titer for that particularstrain of virus before the virus is inactivated for vaccine preparation.Also, depending upon the species, size and condition of the bird beingvaccinated, the immunogenic amount can be varied by the optimizationprocedures taught herein or by procedures known in the art.

The vaccines of the present invention can be used either alone or incombination with a suitable adjuvant. In one embodiment the inventionprovides a vaccine which is protective against avian polyomavirusinfection in a bird which is classified as being a member of thePsittaciformes order, comprising an immunogenic amount of an inactivatedavian polyomavirus, a pharmaceutically acceptable carrier, and anadjuvant which is suitable for use in a bird which is classified asbeing a member of the Psittaciformes order. The term "suitable" is meantto include as an adjuvant, any substance which can be used incombination with the immunogen (e.g., inactivated avian polyomavirus orportion thereof) of the vaccine to augment the immune response withoutproducing adverse side effects in the vaccinated bird. It iscontemplated by the invention that the adjuvants described herein can beutilized in a vaccine against any psittacine pathogen. The adjuvantsdescribed herein can be utilized in any species which is a member of thePsittaciformes order including, but not limited to, the examples ofPsittaciformes cited herein.

In one embodiment, the suitable adjuvant is a long chain polydispersed β(1,4) linked mannan polymer interspersed with O-acetylated groups. Thepresently preferred mannan polymer of the invention is ACEMANNAN. Inanother embodiment, the suitable adjuvant is a deproteinized highlypurified cell wall extract derived from a non-pathogenic strain ofMycobacterium species. A presently preferred Mycobacterium extract isEQUIMUNE.

The vaccine protocol used to administer the immunogenic amount can varydepending upon the species, size, and condition of the bird. The vaccineof the invention is typically administered parenterally, eithersubcutaneously or intramuscularly by injection. Of course, theimmunogenic amount can be given in divided doses or administered atmultiple sites in the bird. Booster immunizations can be given utilizingvaccines containing whole inactivated avian polyomavirus or anyimmunogenic portion thereof.

In one embodiment, the invention specifically provides a compositionwhich produces an anamnestic response against avian polyomavirusinfection in a sensitized bird which is classified as being a member ofthe Psittaciformes order, comprising an anamnestic response inducingamount of a recombinant protein of avian polyomavirus and apharmaceutically acceptable carrier. As used herein, the term"anamnestic response" means a secondary (booster) immune response in asensitized bird. By "sensitized bird" is meant a bird which has beenpreviously been in contact with avian polyomavirus antigen either bynatural exposure to the virus or by vaccination (primary immunization)with avian polyomavirus or an antigenic portion thereof.

The invention also provides the discovery that the VP1 protein of avianpolyomavirus can be utilized to booster the immune response to primaryvaccination against avian polyomavirus in a safe and efficient mannerand with minimal stress to the vaccinated bird. In a presently preferredembodiment, the present invention provides a composition which producesan anamnestic (secondary) response against avian polyomavirus infectionin a sensitized bird which is classified as being a member of thePsittaciformes order, comprising an anamnestic response inducing amountof a recombinant avian polyomavirus VP1 capsid protein and apharmaceutically acceptable carrier. Briefly, the recombinant VP1protein in the presently preferred embodiment was produced in E. coli bycloning the gene that codes for this protein into the pFLAG expressionvector (International Biotechnologies, New Haven, Conn.). The expressedprotein was partially purified by affinity chromatography using ananti-FLAG monoclonal antibody and the composition prepared by adding theprotein to sterile saline (Garcia, A. P., et al., "Diagnosis ofpolyomavirus infection in seedcrackers using DNA in situ hybridization,"J. Assoc. Avian Vet., 1993; In press; Gaskin, 1989).

The other recombinant proteins and polypeptide fragments from polyomavirus can be utilized as an immunogen. Such proteins, or fragmentsthereof, can, for example, be obtained by cloning nucleic acids encodingthe polypeptide in an expression system capable of producing theantigenic polypeptide or fragments thereof.

Given the amino acid sequence of the avian polyomavirus antigen (see,e.g., R. Stoll et al., J. Gen Virology, 1993, 74:229-237), one cansynthesize, using standard peptide synthesis techniques, peptidefragments chosen to be homologous to immunoreactive regions of theantigen and to modify these fragments by inclusion, deletion ormodification of particular amino acid residues in the derived sequences.Thus, synthesis or purification of an extremely large number of peptidesderived from the antigen is possible. Such peptides can be used toimmunize a member of the Psittaciformes order.

The amino acid sequences of the present polypeptides can contain animmunoreactive portion of avian polyomavirus antigen attached tosequences designed to provide for some additional property, such assolubility. The amino acid sequences of an avian polyomavirus antigencan include sequences in which one or more amino acids have beensubstituted with another amino acid to provide for some additionalproperty, such as to remove/add amino acids capable of disulfidebonding, to increase its bio-longevity, alter enzymatic activity, oralter interactions, e.g., at the injection site or with gastric acidityif an oral administration of the vaccine is used. In any case, thepeptide must posses immunogenicity.

Recombinant viral proteins or protein fragments can be tested todetermine their immunogenicity by the methods taught in the examples orby other methods known in the art. Briefly, various concentrations of aputative immunogenically specific fragment are prepared and administeredto a bird and the immunological response (e.g., the production ofantibodies or cell mediated immunity) of the bird to each concentrationis determined. The amount of antigen administered will depend upon thespecies, size and condition of the bird. Thereafter an animal soinoculated with the antigen can be exposed to virulent avianpolyomavirus to test the potential vaccine effect of the specificimmunogenic fragment. The specificity of the putative immunogenicfragment can be ascertained by testing sera, and other fluids orlymphocytes from the inoculated bird, for cross-reactivity with otherclosely related avian polyomaviruses. Once the immunogenicity of a viralfragment is established, the immunogenic amount to be administered to aparticular bird can be determined by optimization procedures as taughtherein and known in the art.

In addition to the E. coli expression vectors herein, there are numerousE. coli expression vectors known to one of ordinary skill in the artuseful for the expression of antigenie avian polyomavirus proteins andpolypeptide fragments. Other microbial hosts suitable for use includebacilli, such as Bacillus subtills, and other enterobacteriaceae, suchas Salmonella, Serratia, and various Pseudomonas species. In theseprokaryotic hosts one can also make expression vectors, which willtypically contain expression control sequences compatible with the hostcell (e.g., an origin of replication). In addition, any number of avariety of well-known promoters will be present, such as the lactosepromoter system, a tryptophan (Trp) promoter system, a beta-lactamasepromoter system, or a promoter system from phage lambda. The promoterswill typically control expression, optionally with an operator sequence,and have ribosome binding site sequences for example, for initiating andcompleting transcription and translation. If necessary an amino terminalmethionine can be provided by insertion of a Met codon 5' and fusedin-frame with the antigen. Also, the carboxy-terminal extension of theantigen can be removed using standard oligonucleotide mutagenesisprocedures.

Additionally, yeast expression can be used. There are several advantagesto yeast expression systems. First, evidence exists that proteinsproduced in a yeast secretion system exhibits correct disulfide pairing.Second, post-translational glycosylation is efficiently carried out byyeast secretory systems. The Saccharomyces cerevisiaepre-pro-alpha-factor leader region (encoded by the MFα-1 gene) isroutinely used to direct protein secretion from yeast. The leader regionof pre-pro-alpha-factor contains a signal peptide and a pro-segmentwhich includes a recognition sequence for a yeast protease encoded bythe KEX2 gene: this enzyme cleaves the precursor protein on the carboxylside of a Lys-Arg dipeptide cleavage-signal sequence. The antigen codingsequence can be fused in-frame to the pre-pro-alpha-factor leaderregion. This construct is then put under the control of a strongtranscription promoter, such as the alcohol dehydrogenase I promoter ora glycolytic promoter. The antigen coding sequence is followed by atranslation termination codon which is followed by transcriptiontermination signals. Alternatively, the antigen coding sequences can befused to a second protein coding sequence, such as Sj26 orβ-galactosidase, used to facilitate purification of the fusion proteinby affinity chromatography. The insertion of protease cleavage sites toseparate the components of the fusion protein is applicable toconstructs used for expression in yeast.

The DNA sequences can be expressed in hosts after the sequences havebeen operably linked to, i.e., positioned to ensure the functioning ofan expression control sequence in an appropriate expression vector.These expression vectors are typically replicable in the host organismseither as episomes or as an integral part of the host chromosomal DNA.Commonly, expression vectors can contain selection markers, e.g.,tetracycline resistance or hygromycin resistance, to permit detectionand/or selection of those cells transformed with the desired DNAsequences (see, e.g., U.S. Pat. No. 4,704,362).

Polynucleotides encoding a variant polypeptide may include sequencesthat facilitate transcription (expression sequences) and translation ofthe coding sequences such that the encoded polypeptide product isproduced. Construction of such polynucleotides is well known in the art.For example, such polynucleotides can include a promoter, atranscription termination site (polyadenylation site in eukaryoticexpression hosts), a ribosome binding site, and, optionally, an enhancerfor use in eukaryotic expression hosts, and, optionally, sequencesnecessary for replication of a vector.

The vaccines and compositions of the invention can include, as notedabove, an effective amount of inactivated avian polyomavirus incombination with a pharmaceutically acceptable carrier and, in addition,may include other medicinal agents, pharmaceutical agents, carriers,adjuvants, diluents, etc. By "pharmaceutically acceptable" is meant amaterial that is not biologically or otherwise undesirable, i.e., thematerial may be administered to an individual along with the selectedcompound without causing any undesirable biological effects orinteracting in a deleterious manner with any of the other components ofthe pharmaceutical composition in which it is contained. Actual methodsof preparing dosage forms are known, or will be apparent, to thoseskilled in this art; for example, see Martin, E. W., Ed., Remington'sPharmaceutical Sciences, latest edition, Mack Publishing Co., Easton,Pa.

Parenteral administration is generally characterized by injection.Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. A morerecently revised approach for parenteral administration involves use ofa slow release or sustained release system, such that a constant levelof dosage is maintained. See, e.g., U.S. Pat. No. 3,710,795.

The present invention also provides a method of preventing avianpolyomavirus infection in a bird which is classified as being a memberof the Psittaciformes order, comprising administering to the bird avaccine comprising an immunogenic amount of an inactivated avianpolyomavirus and a pharmaceutically acceptable carrier. The subject birdof the methods of the invention can be any of the various species ofbirds which are classified as being members of the Psittaciformes orderincluding, but not limited to, the examples cited herein. Specificallyprovided, however, is a method of preventing avian polyomavirusinfection in a bird which is classified as being a member of thePsittaciformes order, comprising administering to the bird a vaccinecomprising an immunogenic amount of an inactivated avian polyomavirusand a pharmaceutically acceptable carrier, wherein the bird is selectedfrom the group consisting of a macaw, an Amazon parrot, a conure, acockatoo, a Pionus Parrot, and an African Grey Parrot.

Also provided is a method of preventing avian polyomavirus infection ina bird from a species which is classified as being a member of thePsittaciformes order, comprising administering to the bird a vaccinecomprising an immunogenic amount of an inactivated avian polyomaviruswhich infects a bird from a different species of the Psittaciformesorder and a pharmaceutically acceptable carrier.

In one embodiment, the vaccine utilized in the methods of the inventionis derived from an isolated avian polyomavirus designated the L4 strain.Given the surprisingly broad species coverage of the L4 strain vaccineas provided herein, other strains of avian polyomavirus isolated fromPsittaciformes bird can be utilized to produce the vaccines of theinvention and utilized in the above methods to cross-protect multiplespecies of Psittaciformes with a single vaccine.

Any of the vaccines and compositions described herein can be utilized inthe methods of the invention, where appropriate, to prevent infectionwith or booster immunity to avian polyomavirus in a subject bird. Forexample, the vaccine utilized in the methods of the invention canfurther comprise an adjuvant suitable for use in a bird which isclassified as a member of the Psittaciformes order. The adjuvant can bea long chain polydispersed β (1,4) linked mannan polymer interspersedwith O-acetylated groups such as, e.g., ACEMANNAN (CarringtonLaboratories, Dallas, Tex.) or a deproteinized highly purified cell wallextract derived from a non-pathogenic strain of Mycobacterium speciessuch as, e.g., EQUIMUNE (Vetrepharm Research Inc., Athens, Ga.).

In the methods described herein, the administering step is typicallypreformed by parenteral administration, i.e., subcutaneous orintramuscular injection of the vaccine into the subject bird. Theimmunogenic amount of vaccine utilized in the methods of the inventionis the same as that provided for in the vaccines of the invention.Specifically, the immunogenic amount of the inactivated avianpolyomavirus corresponds to a titer of between 10⁴.5 TCID₅₀ and 10⁷TCID₅₀ for the avian polyomavirus before inactivation but especiallyabout 10⁵.8 TCID₅₀.

The methods of the invention can further comprise the step ofadministering at least one booster vaccine to the bird. One or morebooster inoculations are typically administered at bi-weekly intervals.The booster vaccine can be any of the vaccine preparations contemplatedherein. However, a preferred embodiment of the invention provides amethod of preventing avian polyomavirus infection in a bird which isclassified as being a member of the Psittaciformes order, thecomposition comprising administering to the bird a vaccine comprising animmunogenic amount of an inactivated avian polyomavirus and apharmaceutically acceptable carrier. After the initial inoculation, atleast one booster vaccine is administered to the bird. The boostervaccine is a composition which produces an anamnestic response againstavian polyomavirus infection in a sensitized bird which is classified asbeing a member of the Psittaciformes order. The booster comprises ananamnestic response inducing amount of a recombinant protein of avianpolyomavirus and a pharmaceutically acceptable carrier. The boostervaccine can be comprised of any recombinant protein derived from avianpolyomavirus or an immunogenic polypeptide fragment thereof. In oneembodiment, the recombinant protein is the VP1 capsid protein.

Briefly, the first booster vaccine can be administered to the subjectbird about two weeks following primary inoculation. If desired, a secondbooster can be administered in about two weeks.

A recombinant protein such as the VP1 protein produces a specificantibody response in the animal to only a portion of the virus.Secondary response to a specific immunogenic protein greatly reduces therisks associated with booster vaccination. Reaction to the booster istherefore milder yet sufficiently immunogenic to boost the bird'sprimary immunity to the virus. In addition to being safer and lessstressful, booster vaccines derived from recombinant proteins are moreeconomical to manufacture.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

EXAMPLES Example I: Inactivated Avian Polyomavirus Vaccine Material andMethods

Virus: A stock strain of avian polyomavirus (passage level 6), recoveredoriginally from infected Budgerigars (Melopsittacus undulatus), wasgrown in chicken embryo fibroblasts in M199 tissue culture mediumsupplemented with 5% calf serum and was used for all the experimentsdescribed in this study. Flasks of infected cells were grown for 7 daysand were then frozen and thawed three times. The suspended cells werehomogenized using a glass bead homogenizer with 0.1 mm beads accordingto the manufacturer's recommendations (Bead Beater, Biospec Products,Bartlesville, Okla.). Beads and cellular debris were separated from thesupernatant by centrifugation (300×g for 5 minutes). Thevirus-containing supernatant (50 ml) was serially diluted in aMICROTITER plate containing a 24-hour monolayer of primary chickenembryo fibroblasts. This preparation had an infectivity titer of 10⁴.3TCID₅₀ per ml.

The virus preparation was purified by isopycnic centrifugation.Initially, the cellular suspension was clarified by centrifugation(7000×g for 45 minutes). The clarified supernatant was layered over 45%(w/v) sucrose in phosphate-buffered saline solution (PBSS, pH 7.2) andcentrifuged (140,000×g, 2 hours, 4° C.) to obtain crude viral pellets(Beckman L8-80M Ultracentrifuge, type 35 rotor, Beckman Instruments,Palo Alto, Calif.). The virus was resuspended in PBSS, adjusted to 1.34g/ml with cesium chloride, and centrifuged to equilibrium (270,000×g, 16hours, 20° C.) (Beckman L8-80M Ultracentrifuge, type 70.1 rotor, beckmanInstruments, Palo Alto, Calif.). The gradient was collected with adensity gradient fractionator (Density Gradient Fractionator, Isco,Lincoln, Nebr.). Fractions containing purified virus were dialyzedagainst PBSS and stored at -20° C. Fifty microliters of the virussuspension was serially diluted in a MICROTITER plate containing a24-hour monolayer of primary chicken embryo fibroblasts. Fractionscontaining purified virus were dialyzed against PBSS and stored at - 20°C. The cesium chloride purified virus preparation had an infectivitytiter of 10⁵.8 TCID₅₀ per ml.

Virus neutralizing antibody assay: Polyomavirus neutralizing antibodytiters were determined according to published procedures (Lukert, P. D.,"Budgerigar fledgling disease," Purchase H. G., et al., Ed., ALaboratory Manual for the Isolation and Identification of AvianPathogens, Kennet Square: Am. Assoc. Avian Pathol., 1989:106-107) withthe exception that the chicken embryo fibroblasts were fixed with 95%ethanol and stained with crystal violet to detect CPE (cytopathiceffect). Antibody titers were expressed as the reciprocal of the serumdilution that protected the chicken embryo fibroblasts from detectablecytopathic effects.

Vaccine preparation: The purified virus used to produce the vaccine hada titer of 10⁵.8 TCID₅₀ per ml before inactivation. The purified viruswas inactivated by adding a sufficient quantity of g-propiolactone tocreate a solution with a final concentration of 0.1% β-propiolactone.The solution was incubated with constant mixing at 37° C. for 4 hours.The mixture was then placed in a refrigerator at 4° C. overnight. Thispreparation was evaluated to demonstrate that the virus had beeninactivated by placing 50 μl of the β-propiolactone treated virus in a24-hour monolayer of chicken embryo fibroblasts. A control vaccine wasproduced by making a PBSS solution that contained a final concentrationof 0.1% β-propiolactone.

Vaccination protocol: Six 30-to 45-day-old Blue and Gold Macaw (Araararauna) chicks from an aviary with no previous history of clinicalavian polyomavirus infections were used in this study. The chicks weredivided randomly into 3 pairs and housed in groups of 2. The vaccinatedand unvaccinated chicks were maintained in separate locations to reducethe possibility of inadvertent viral exposure in the unvaccinatedchicks. Blood was collected by jugular venipuncture from all chicks ondays 0, 10, 20, 30 and 37. On days 10, 20 and 30, two groups of chickswere vaccinated subcutaneously with 50 μl of inactivated polyomavirussuspension emulsified in 150 μl of an oil adjuvant. The third group ofchicks was vaccinated on the same days subcutaneously withβ-propiolactone mixed in 50 μl of PBSS emulsified in 150 μl of an oiladjuvant. The chicks were examined immediately after inoculation andfour times each day to determine whether vaccination caused anyimmediate or delayed local or systemic reactions.

Challenge: All chicks were challenged with live virus on days 37, 39 and52. The in vitro infectivity of the challenge inoculum was confirmed bydemonstrating characteristic cytopathic effects when a 50-μl sample ofthe challenge inoculum was placed on a 24-hour monolayer of chickenembryo fibroblasts grown in MICROTITER plates as described above. Ondays 37 and 39, the chicks were administered 50 μl of virus suspensioncontaining 10³.3 TCID₅₀ of live virus. On day 52, the chicks were given10⁴.5 TCID₅₀ of live virus. On each day of challenge, two of thevaccinated chicks received 50 μl of virus preparation by the oral andintracloacal routes; another two 50 μl of virus preparation byintramuscular inoculation. One of the unvaccinated control birdsreceived 50 μl of virus preparation by the oral and intracloacal routes;the other 50 μl of virus preparation by intramuscular inoculation. Bloodwas collected from the chicks by jugular venipuncture on days 37, 46, 59and 68. The blood samples were allowed to clot, and the serum wascollected and used to determine virus-neutralizing antibody titers asdescried above. The chicks were examined four times a day for clinicalchanges suggestive of an active infection or adverse response tochallenge.

DNA probe detection of polyomavirus nucleic acid: Whole blood samplescollected in sodium heparin (20 μl heparin per ml of blood) wereobtained on days 37, 46, 59 and 68. Samples were processed for detectionof polyomavirus nucleic acid using amplification procedures andviral-specific DNA probes according to a modification of publishedprocedures (Niagro, F. D., et al., "Use of polymerase chain reaction fordetection of BFD in suspect birds," Proc. Assoc. Avian Vet., Phoenix,1990, 25-37). Cloacal swabs were collected from the chicks on days 10,20, 37 and daily thereafter. The swabs were collected before feedingeach morning. These swabs were processed for detection of polyomavirusnucleic acid, as described previously (Davis et al., 1981).

Results

None of the chicks used in this study had detectable levels ofpolyomavirus neutralizing (VN) antibodies at the beginning of the study.The virus used for vaccination did not induce detectable cytopathiceffects in a monolayer of chicken embryo fibroblasts after 7 days ofincubation confirming that it had been inactivated following theaddition of β-propiolactone. The vaccinated chicks developed atransitory polyuria (24 hrs) the day after the first vaccination andwere considered to be slightly lethargic for a 24-to-48 hour periodstarting two days after each vaccination. The VN antibody titers thatdeveloped in the chicks vaccinated with β-propiolactone-treated PBSS(controls) or with inactivated avian polyomavirus are listed in Table 1.The chicks inoculated with β-propiolactone-treated PBSS emulsified in anoil adjuvant remained seronegative throughout the prechallenge period.The first increase in VN titers in the chicks vaccinated withinactivated antigen was detected 20 days after the initial vaccination.By the 37th day after the initial vaccination, the VN titers ranged from20 to 40, with a geometric mean titer of 28.

All of the chicks remained clinically normal following challenge withlive virus. The unvaccinated chicks had a 6-to-7 fold increase in VNantibody titer, suggesting that an active infection had occurred. Noneof the vaccinated chicks had a significant increase (greater than 4fold) in VN antibody titer following challenge (Table 2). All of thechicks vaccinated with oil-adjuvanted vaccine developed lesions at eachsite of subcutaneous inoculation. Two of the birds developed moderatereactions (subcutaneous mass<0.5 cm in diameter) and two developedsevere reactions (subcutaneous mass>0.5 cm in diameter with skinnecrosis).

Using DNA probes, viral nucleic acid could not be demonstrated incloacal swabs collected from any of the chicks prior to challenge (day37). Viral nucleic acid was detected in cloacal swabs from theunvaccinated chicks exposed to live virus by the oral and intracloacalroutes on days 1 and 2 after challenge. Viral nucleic acid was detectedin cloacal swabs from the unvaccinated chicks exposed to live virus bythe intramuscular route on days 2 and 3 after challenge. Using the samedetection technique, viral nucleic acid could not be demonstrated incloacal swabs from the vaccinated chicks at any time during the study.The DNA probes failed to demonstrate the presence of viral nucleic acidin the amplified products from any of the whole blood samples tested.

Discussion

The vaccine of the present invention elicited polyomavirus neutralizingantibodies in all the vaccinates. The induced immunologic responseprotected the vaccinates from subsequent challenge with live virus.Although the sample size was small, all of the vaccinated chicks wereresistant to infection, whereas the unvaccinated chicks became infected,demonstrating that the inactivated vaccine used in this study waseffective.

Viral replication did not appear to occur in the vaccinated chicksbecause there was no significant change (greater than 4 fold increase)in the VN antibody titers in these birds following challenge. Anincrease in titers would have indicated an active infection with anassociated anamnestic response. Although the detectable immunologicresponse in the form of VN antibodies was low (geometric mean titer,28), the vaccinated chicks were resistant to challenge demonstratingthat a protective immunogenic response occurred following vaccination.

Example II: A Suitable Adjuvant for Psittaciformes

Four adjuvants (mineral oil, E-3, EQUIMUNE, and ACEMANNAN) wereevaluated to determine their effect on the immunogenicity of inactivatedpolyomavirus and the degree of local reactions that they induced wheninjected into a group of mixed species of Psittaciformes. All fouradjuvant-antigen combinations were found to stimulate the production ofanti-polyomavirus neutralizing antibodies. All of the birds vaccinatedwith the oil-adjuvanted vaccine developed abscesses or open draininglesions at the sites of the subcutaneous inoculation. Three of the eightbirds vaccinated with E-3 adjuvanted antigen died following the secondvaccination. The EQUIMUNE-adjuvanted antigen induced small ulceratedlesions with subsequent scab formation in one of the three vaccinates.The ACEMANNAN adjuvanted antigen was associated with the mildestreaction which was limited to hyperemia at the site of initialsubcutaneous inoculation in one of the seven vaccinates.

A group of adult psittacine birds with representatives from 8 differentgenera was used to determine whether any of four commercially availableadjuvants mixed with inactivated avian polyomavirus would induce theformation of anti-polyomavirus neutralizing antibodies without causingunacceptable adverse systemic or local reactions.

Materials and Methods

Birds: The study population included 26 adult psittacine birds: 2Umbrella Cockatoos (Cacatua alba), 4 African Grey Parrots (Psittacuserithacus erithacus), 4 Ducorp's Cockatoos (Cacatua ducorps), 1Sulphur-crested Cockatoo (Cacatua sulphurea), 1 Goffin's Cockatoo(Cacatua goffini), 1 Red-fronted Conure (Aratinga wagleri wagleri), 1Pionus Parrot (Pionus maximillani), 1 Sun Conure (Aratingasolstitialis), 1 Yellow-Naped Amazon Parrot (Amazona ochrocephalaauropalliata), 5 White-bellied Caiques (Pionites leucogasterleucogaster), 1 Golden Conure (Aratinga guarouba), 1 Green Conure(Aratinga holochlora rubritorquis), 1 Blue-crowned Conure (Aratingaacuticaudata acuticaudata), 1 Ringed-neck Parakeet (Psittacula kramerikrameri) and 1 Blue and Gold Macaw (Ara ararauna). Nine of the birdswere known to be carriers of avian polyomavirus because of intermittentdetection of viral nucleic acid in their excrement using DNA probes andthe detection of sustained high polyomavirus-neutralizing antibodytiters for over one year (Niagro, F. D., et al., "Avian polyomavirus:Discordance between neutralizing antibody titers and viral shedding inan aviary," Proc. Assoc. Avian Vet., 1991, 22-26; Gaskin, 1988).

Virus: A stock strain of Budgerigar fledgling disease virus, isolatedoriginally from infected Budgerigars (Melopsittacus undulatus), wasgrown in chicken embryo fibroblasts in M199 with 5% calf serum. Infectedcells were incubated 5 to 7 days. The culture flasks were frozen andthawed three times, and the contents were pooled. Freeze-thawedsuspensions were centrifuged (500×g for 10 minutes) at 4° C. to removecellular debris and the supernatant was used as the stock virus. Theinfectivity titer of the stock virus was determined by making ten-foldserial dilutions in M199 with fetal calf serum. Fifty microliters ofeach dilution (10⁻³ through 10⁻⁸) was inoculated in replicates of eightper dilution into flat bottom 96-well MICROTITER plates. To each well,200 ul of packed chicken embryo fibroblasts (in M199 with 5% fetal calfserum) was then added in a ratio of 1:200, respectively. Plates wereincubated for 6 days, fixed with 95% ethanol, stained with crystalviolet, and evaluated for cytopathic effects (CPE). The Spearman-Karbermethod (Villegas, P., Purchase, G. H., "Titration of biologicalsuspensions," Hitchner, S. B., et al., Ed., Isolation and Identificationof Avian Pathogens, Endwell: Creative Publishing Co., 1980) was used tocalculate the TCID₅₀ of the stock virus preparation. The infectivitytiter of the virus preparation used for vaccination on days 0 and 14 wasapproximately 10⁷ TCID₅₀ per ml and the infectivity tiler of the viruspreparation used for all other vaccinations was approximately 10⁹ TCID₅₀per ml.

Vaccine preparation: The virus used for vaccination was produced asdescribed above except that the growth media was changed to M199 withoutcalf serum 24 hours after the cell cultures were inoculated. The viruswas inactivated by adding β -propiolactone. After 1 ml ofβ-propiolactone (95% solution) was added to 500 ml of virus preparation,the pH was adjusted to 7.0 by the addition of 1N sodium hydroxide(NaOH). This mixture was stirred constantly at room temperatureovernight. One microliter of β-propiolactone (95% solution) was thenadded to 1000 ml of virus preparation and the pH adjusted to 7.0 by theaddition of 1N NaOH. This mixture was maintained at room temperature andstirred constantly for 2 hours. The β-propiolactone was then neutralizedby the addition of 1 ml of 0.2M sodium thiosulfate per 100 ml of virussuspension. The mixture was stored at 4° C. until used. This preparationwas assayed for infectivity to insure complete virus inactivation byplacing 50 μl of the β-propiolactone treated virus preparation onchicken embryo fibroblasts as described above.

Two birds were vaccinated with 500 μl of β-propiolactone treated virus(Table 5). Twenty-two birds were vaccinated with β-propiolactone treatedvirus mixed with one of four adjuvants. The adjuvants used included:ACEMANNAN, a long chain polydispersed β (1,4) linked mannan polymerinterspersed with O-acetylated groups (Carrington Laboratories, Dallas,Tex.); EQUIMUNE, a deproteinized highly purified cell wall extractderived from non-pathogenic strains of Mycobacterium species (VetrepharmResearch Inc., Athens, Ga.) and E3, an immunomodulator derived from thecell wall of E. coli (Immvac Inc., Colombia, Miss.). The β-propiolactonetreated virus was mixed with each of the adjuvants so that the finalsuspension for inoculation contained 500 μl of inactivated virus. Forthe oil-adjuvanted vaccine, each dose represented a volume of 300 μl ofmineral oil that had been mixed with 500 μl of β-propiolactone treatedvirus. For the ACEMANNAN and E3 vaccines, 100 μl of each adjuvant wasmixed with 500 μl of β-propiolactone treated virus. For the EQUIMUNEvaccine, 50 μ l of adjuvant was mixed with 500 μl of β-propiolactonetreated virus.

Two birds were vaccinated with recombinant VP1 protein emulsified inmineral oil. Recombinant VP1 protein was produced in E. coli by cloningthe gene that codes for this protein into the pFLAG expression vector(International Biotechnologies, New Haven, Conn.). The expressed proteinwas partially purified by affinity chromatography using an anti-FLAGmonoclonal antibody (Lin, K. H., Cheng, S. Y., "An efficient method topurify active eukaryotic proteins from the inclusion bodies in E. coli,"Biotechniques, 1991, 11:748-753). Twenty-five milligrams of protein (asdetermined using the Bradford reagent) (Bradford, M. M., "A rapid andsensitive method for the quantitation of microgram quantities of proteinutilizing the principle of protein-dye binding," Anal. Biochem. 1976,72:248-254) was added to 500 μl of sterile saline solution and mixedwith 300 μl of mineral oil.

Vaccination protocol: All injections and blood collections wereperformed with the birds under isoflurane anesthesia. Blood wascollected by jugular venipuncture from each vaccinate on days 0, 14, 28,42, 49 and 63. After blood collection, contour feathers were removedfrom the caudal proventer region and the birds were inoculatedsubcutaneously on days 0, 14 and 28. Alternating sides of the proventerregion were used for each inoculation to help distinguish which of theinjections was associated with a reaction.

The birds were divided into two groups on day 42 to determine the grossor clinically detectable systemic affects of repeated exposure andintramuscular inoculation with the two most promising adjuvants. Halfthe birds were vaccinated with ACEMANNAN-antigen and half werevaccinated with EQUIMUNE-antigen by deep intramuscular injection intothe pectoral muscle on days 42 and 49. Preparation of the vaccine forintramuscular inoculation was the same as described above.

Grading reactions: The birds were examined immediately after inoculationand daily to determine whether the vaccine caused any immediate ordelayed local or systemic reactions. The injection sites were observedand palpated at each booster time for the presence of hyperemia,discoloration of the skin, swelling, thickening of the skin, necrosis,and abscess or scab formation. The size and location of each reactionwas recorded according to the scheme: 0=no reaction; 1=slight reaction(hyperemia, skin discoloration); 2=mild reaction (small scab formation,thickening of the skin); 3=moderate reaction (subcutaneous mass<0.5 cmin diameter or<0.5 cm area of necrosis); 4=severe reaction (>0.5 cmsubcutaneous mass, or>0.5 cm area of necrosis).

Virus neutralizing antibody assay: To detect virus neutralizingantibodies, blood collected from each bird was placed in sodium EDTA,allowed to settle, and the plasma was separated by centrifugation. Theplasma was stored at -20° C. until assayed. Polyomavirus neutralizingantibody titers were determined as previously described except that thechicken embryo fibroblasts were fixed with 95% ethanol and stained withcrystal violet to detect CPE.1 Antibody titers were expressed as thereciprocal of the serum dilution that protected the chicken embryofibroblasts from detectable cytopathic effects as detected by reducedstaining with crystal violet. Rabbit anti-polyomavirus antibodies with aVN titer of 1:1024 and normal chicken serum with a VN titer of<1:2 wereused as positive and negative controls, respectively.

Results

Cytopathic effects (CPE) were not observed in chicken embryo fibroblastsinoculated with β-propiolactone treated virus indicating that the virusused for vaccination in this study had been inactivated. Eighty-sixpercent (18 of 21) of the birds vaccinated with inactivated avianpolyomavirus seroconverted (greater than four fold increase in VNantibody liter with the resulting titer>1:10) by two weeks after thethird vaccination (day 42, Table 3). Three birds (#1, #22 and #23) diedfollowing the second booster inoculation with the E3-adjuvanted vaccineand were unavailable for titer calculations after day 28. Two otherbirds (#20 and #25) that did not seroconvert following subcutaneousvaccination were from the E3 adjuvanted vaccine group and did notreceive a third booster vaccination. However, these latter birds alsofailed to seroconvert when vaccinated by the intramuscular route withantigen mixed with either ACEMANNAN or EQUIMUNE (Table 4). One-hundredpercent (13 of 13) of the birds vaccinated with antigen alone or antigenmixed with mineral oil or ACEMANNAN seroconverted by the second weekafter the third inoculation (day 42, Table 3).

For each vaccinate, the highest VN antibody tiler detected by the secondweek after the third vaccination (day 42) in the oil-adjuvanted groupranged from 1:16 to 1:131,072 (geometric mean titer GMT=724), in theantigen only group ranged from 1:16 to 1:1024 (GMT=128), in the E3 groupranged from 1:4 to 1:16,384 (GMT=54), in the ACEMANNAN group ranged from1:16 to 1:8192 (GMT=256) and in the EQUIMUNE group ranged from 1:8 to1:131,072 (GMT=2580). One of two birds that were seropositive at thestart of the study (#10 and #14) did seroconvert following vaccinationwith recombinant VP1 (rVP1) mixed in an oil adjuvant. These birds wereinoculated with this vaccine to determine whether the rVP1 protein wasimmunogenic.

Of the vaccinates that were seropositive (VN antibody titer>1:10) priorto vaccination, 100% (7 of 7, bird #1 died following the secondvaccination and is not included) seroconverted by two weeks after thesecond vaccination (day 28, Table 5). Of the birds that wereseronegative (VN antibody titer<1:10) prior to vaccination, 71% (10 of14) of these initially seronegative birds seroconverted by two weeksafter the second vaccination (day 28, Table 5).

The use of E3 as an adjuvant was discontinued when three vaccinates diedwithin two days of each other after the second inoculation. In thesebirds, necropsy findings considered relative to the vaccinationprocedure included: focal severe necrotizing dermatitis and myositis atthe site of vaccination (all three birds); myeloid hyperplasia in thebone marrow suggestive of a heterophilic response to tissue necrosis (1bird); Shwartzman reaction in the kidney (1 bird); and diffuseGram-negative septicemia (1 bird).

Birds at the beginning of the study that had positive antibody titersdeveloped higher VN antibody titers by the second week after the thirdsubcutaneous vaccination than those birds that were consideredseronegative at the beginning of the study (Table 5). However, therelative increase in titers was virtually the same, with a 4.2-foldincrease in antibody titer in the seronegative group and a 4.1-foldincrease in antibody titer in the seropositive group. The highest titersmeasured by the second week after the third subcutaneous vaccination(day 42) in the seropositive group ranged from 1:512 to 1:131,072(GMT=8192, bird #1 not included) while the highest titers in theseronegative group ranged from 1:4 to 1:1024 (GMT=35, birds #22 and #23not included).

All vaccination-induced lesions were allowed to resolve withouttreatment to observe the natural progression of the lesions (Table 6).None of the birds developed lesions that were considered to be inducingundue distress or pain. The appetites and attitudes of the birdsremained normal throughout the study except for one caique (#14) thatexhibited a 3-day period of depression and lethargy after the secondvaccination. This bird recovered uneventfully.

All of the birds (6 of 6) vaccinated with oil-adjuvanted vaccinedeveloped some type of lesion after subcutaneous inoculation. Thesereactions were classified as moderate to severe. Half of the birds inthis group that developed lesions were seronegative at the start of thestudy and half of the birds were seropositive. One of the birds (#21) inthe non-adjuvanted vaccine group developed a mild reaction characterizedby hyperemia and thickening of the skin. Reactions in the survivingmembers of the E3 group were considered mild. The cutaneous andsubcutaneous lesions in the birds that died were considered clinicallymild, but histologically severe. The reactions in the EQUIMUNE groupwere considered mild. ACEMANNAN was considered the least reactiveadjuvant, with one vaccinate developing a slight reaction (hyperemia)following the initial vaccination. None of the birds vaccinated by theintramuscular route with ACEMANNAN or EQUIMUNE developed visible orpalpable reactions. No difference was noted with respect to reactions inthe birds that were either seropositive or seronegative at the start ofthe study.

Discussion

Some of the birds used in this study were considered to be polyomaviruscarriers for two reasons; the demonstration of viral nucleic acid incloacal swabs using DNA amplification procedures and demonstration ofsustained high antibody titers (over one year of sustained high titers)(Niagro et al., 1991; Gaskin, 1988). Birds that were seropositive wereused in this study to determine whether an adjuvanted inactivated avianpolyomavirus vaccine would cause any adverse reactions in birds thatalready had a VN titer to polyomavirus from a previous or sustainedinfection. The initially seropositive birds used in this study developedno observable adverse reactions following vaccination that were notattributable to the adjuvant. Given the seroprevalence of the disease incompanion birds and the frequency of subclinical carriers (Niagro etal., 1991; Gaskin, 1988; Jacobson et al., 1984; Clubb and Davis, 1984;Wainwright et al., 1987), it is noteworthy that an inactivated vaccineintended for commercial release did not cause adverse reactions invaccinates that were seropositive prior to vaccination.

The birds in this study were initially vaccinated by the subcutaneousroute, and several adjuvants were used. After the initial series ofsubcutaneous vaccinations, the birds were vaccinated by theintramuscular route using either ACEMANNAN or EQUIMUNE as adjuvants.Repeated exposure to these vaccines was intended to determine whetherunacceptable local or clinically detectable systemic reactions mightoccur following frequent vaccination with the most promising adjuvants.

Reactions at the injection site varied with the type of adjuvant. Theoil-adjuvanted reactions were moderate to severe, whereas the reactionswith the other adjuvants were slight, mild, or undetectable. Theoil-adjuvanted vaccine caused lesions that included severe granulomaformation and necrosis. In all, each bird was exposed to a minimum of 5injections with an adjuvanted antigen (except for the E3 group thatreceived 4 inoculations) over a 7-week period. The ACEMANNAN andEQUIMUNE adjuvants did not cause unacceptable reactions even followingthis high frequency of exposure to the adjuvant antigen mixture.Considering the ease of administration and the mild post-vaccinationalreactions, a vaccine that contains an alternative adjuvant to oil wouldappear to be the most efficacious for use with avian polyomavirus inpsittacine birds.

Although the present process has been described with reference tospecific details of certain embodiments thereof, it is not intended thatsuch details should be regarded as limitations upon the scope of theinvention except as and to the extent that they are included in theaccompanying claims.

                  TABLE 1                                                         ______________________________________                                        Virus-neutralizing antibody titers in a group of chicks                       on days 10, 20 and 30 after vaccination with                                  β-propiolactone-inactivated avian polyomavirus                           β-propiolactone                                                          treated PBSS                                                                  (virus-free    Inactivated avian                                              inoculum)      polyomavirus vaccine                                           Day   Bird 1  Bird 2   Bird 3                                                                              Bird 4 Bird 5                                                                              Bird 6                              ______________________________________                                        0     0       0        0     0      0     0                                   10    0       0        0     0      0     0                                   20    0       0        0     0      0     0                                   30    0       0        10    0      10    0                                   37    0       0        40    20     20    40                                  ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Virus-neutralizing antibody titers in a group                                 of chicks overtime after viral challenge                                      with viable avian polyomavirus on days 37, 39 and 52                          β-propiolactone                                                          treated PBSS                                                                  (virus-free                                                                   inoculum)                                                                     Bird 1   Bird 2  Inactivated avian polyomavirus vaccine                            oral    IM      Bird 3 Bird 4 Bird 5 Bird 6                                   chal-   chal-   oral   IM     oral   IM                                  Day  lenge   lenge   challenge                                                                            challenge                                                                            challenge                                                                            challenge                           ______________________________________                                        37   0       0       40     20     20     40                                  46   0       20      40     20     10     40                                  59   640     320     80     40     20     20                                  68   640     640     80     40     20     20                                  ______________________________________                                    

                                      TABLE 3                                     __________________________________________________________________________    Summary of reciprocal VN antibody titers                                      in psittacine birds vaccinated by the subcutaneous                            route with various antigen-adjuvant combinations                                            Bird        Days Post Initial Vaccination                       Species       #   Adjuvant                                                                              0   14   28    42                                   __________________________________________________________________________    Umbrella Cockatoo                                                                           8   Oil     <2  128  256   1024                                 Ducorp's Cockatoo                                                                           11  Oil     <2  4    4     16                                   Green Conure  16  Oil     --  2048 32,768                                                                              131,072                              African Grey Parrot                                                                         19  Oil     <2  128  128   64                                   Sulphur-crested Cockatoo                                                                    9   Antigen Only                                                                          <2  1024 512   256                                  African Grey Parrot                                                                         21  Antigen Only                                                                          <2  16   16    8                                    Caique        1   E3      512 1024 *     *                                    Sun Conure    4   E3      512 16,384                                                                             16,384                                                                              16,384                               Amazon Parrot 18  E3      2   4    16    32                                   Ducorp's Cockatoo                                                                           20  E3      2   4    4     2                                    African Grey Parrot                                                                         22  E3      <2  2    *     *                                    Golden Conure 23  E3      <2  64   *     *                                    Goffin's Cockatoo                                                                           24  E3      <2  64   32    64                                   Pionus Parrot 25  E3      <2  2    4     4                                    Red-fronted Conure                                                                          10  VP1 + Oil                                                                             64  256  256   512                                  Caique        14  VP1 +  Oil                                                                            2048                                                                              4096 512   1024                                 Ringneck Parakeet                                                                           2   Acemannan                                                                             <2  16   32    16                                   Blue and Gold Macaw                                                                         5   Acemannan                                                                             128 2048 1024  2048                                 Ducorp's Cockatoo                                                                           6   Acemannan                                                                             <2  4    16    32                                   Caique        7   Acemannan                                                                             1024                                                                              4096 8192  2048                                 Blue-crowned Conure                                                                         13  Acemannan                                                                             <2  4    16    16                                   Umbrella Cockatoo                                                                           15  Acemannan                                                                             512 4096 8192  4096                                 African Grey Parrot                                                                         17  Acemannan                                                                             4   16   32    16                                   Ducorp's Cockatoo                                                                           3   Equimune                                                                              <2  8    8     8                                    Caique        12  Equimune                                                                              2048                                                                              32,768                                                                             131,072                                                                             65,536                               Caique        26  Equimune                                                                              1024                                                                              4096 16,384                                                                              16,384                               __________________________________________________________________________     *Bird died                                                               

                  TABLE 4                                                         ______________________________________                                        Summary of the reciprocal VN antibody titers that occurred                    following intramuscular vaccination with antigen mixed                        with either Acemannan or Equimune adjuvant                                                            Days Post Initial                                              Bird           Vaccination                                           Species    #      Adjuvant  42     49    63                                   ______________________________________                                        Ringneck   2      Acemannan 16     32    16                                   Parakeet                                                                      Blue and Gold                                                                            5      Acemannan 2048   1024  1024                                 Macaw                                                                         Ducorp's   6      Acemannan 32     32    4                                    Cockatoo                                                                      Caique     7      Acemannan 2048   8192  4096                                 Umbrella   8      Acemannan 1024   1024  1024                                 Cockatoo                                                                      Sulphur-crested                                                                          9      Acemannan 256    1024  512                                  Cockatoo                                                                      Ducorp's   11     Acemannan 16     32    64                                   Cockatoo                                                                      Blue-crowned                                                                             13     Acemannan 16     32    32                                   Conure                                                                        Umbrella   15     Acemannan 4096   8192  4096                                 Cockatoo                                                                      African Grey                                                                             17     Acemannan 16     1024  512                                  Parrot                                                                        Ducorp's   20     Acemannan 2      4     16                                   Cockatoo                                                                      Goffin's Cockatoo                                                                        24     Acemannan 64     128   256                                  Ducorp's   3      Equimune  8      8     32                                   Cockatoo                                                                      Sun Conure 4      Equimune  16,384 16,384                                                                              8192                                 Red-fronted                                                                              10     Equimune  512    2048  4096                                 Conure                                                                        Caique     12     Equimune  65,536 16,384                                                                              65,536                               Caique     14     Equimune  1024   2048  32,768                               Green Conure                                                                             16     Equimune  131,072                                                                              32,768                                                                              16,384                               Amazon Parrot                                                                            18     Equimune  32     512   512                                  African Grey                                                                             19     Equimune  64     256   256                                  Parrot                                                                        African Grey                                                                             21     Equimune  8      32    64                                   Parrot                                                                        Pionus Parrot                                                                            25     Equimune  4      8     16                                   Caique     26     Equimune  16,384 16,384                                                                              4096                                 ______________________________________                                    

                                      TABLE 5                                     __________________________________________________________________________    Summary of reciprocal VN antibody titers in birds vaccinated by the           subcutaneous route when divided into birds that were seronegative or          seropositive prior to vaccination                                                             Bird        Days Post Initial Vaccination                     Species         #   Adjuvant                                                                              0   14   28    42                                 __________________________________________________________________________    SERONEGATIVE GROUP                                                            Ringneck Parakeet                                                                             2   Acemannan                                                                             <2  16   32    16                                 Ducorp's Cockatoo                                                                             3   Equimune                                                                              <2  8    8     8                                  Ducorp's Cockatoo                                                                             6   Acemannan                                                                             <2  4    16    32                                 Umbrella Cockatoo                                                                             8   Oil     <2  128  256   1024                               Sulphur-crested Cockatoo                                                                      9   Antigen Only                                                                          <2  1024 512   256                                Ducorp's Cockatoo                                                                             11  Oil     <2  4    4     16                                 Blue-crowned Conure                                                                           13  Acemannan                                                                             <2  4    16    16                                 African Grey Parrot                                                                           17  Acemannan                                                                             4   16   32    16                                 Amazon Parrot   18  E3      2   4    16    32                                 African Grey Parrot                                                                           19  Oil     <2  128  128   64                                 Ducorp's Cockatoo                                                                             20  E3      2   4    4     2                                  African Grey Parrot                                                                           21  Antigen Only                                                                          <2  16   16    8                                  African Grey Parrot                                                                           22  E3      <2  2    *     *                                  Golden Conure   23  E3      <2  64   *     *                                  Goffin's Cockatoo                                                                             24  E3      <2  64   32    64                                 Pionus Parrot   25  E3      <2  2    4     4                                  SEROPOSITIVE GROUP                                                            Caique          1   E3      512 1024 *     *                                  Sun Conure      4   E3      512 16,384                                                                             16,384                                                                              16,384                             Blue and Gold Macaw                                                                           5   Acemannan                                                                             128 2048 1024  2048                               Caique          7   Acemannan                                                                             1024                                                                              4096 8192  2048                               Caique          12  Equimune                                                                              2048                                                                              32,768                                                                             131,072                                                                             65,536                             Umbrella Cockatoo                                                                             15  Acemannan                                                                             512 4096 8192  4096                               Green Conure    16  Oil     --  2048 32,768                                                                              131,072                            Caique          26  Equimune                                                                              1024                                                                              4096 16,384                                                                              16,384                             __________________________________________________________________________     *Bird died                                                               

                                      TABLE 6                                     __________________________________________________________________________    Summary of post-vaccinational reactions in a                                  group of mixed species psittaciformes                                                       Bird        Days Post Initial Vaccination                       Species       #   Adjuvant                                                                              0  14   28    42    49    63                        __________________________________________________________________________    Umbrella Cockatoo                                                                           8   Oil     0  4    4     3     3     3                                                      1st site                                                                           2nd site                                                                            bilateral                                                                           bilateral                                                                           bilateral                 Ducorp's Cockatoo                                                                           11  Oil     0  0    0     2     2     0                                                                 2nd site                                                                            2nd site                        Green Conure  16  Oil     0  2    3     2     2     2                                                      1st site                                                                           2nd site                                                                            2nd site                                                                            2nd site                                                                            2nd site                  African Grey Parrot                                                                         19  Oil     0  2    4     3     3     2                                                      1st site                                                                           2nd site                                                                            bilateral                                                                           bilateral                                                                           bilateral                 Red-fronted Conure                                                                          10  VP1 + Oil                                                                             0  3    4     4     3     3                                                      1st site                                                                           2nd site                                                                            bilateral                                                                           bilateral                                                                           bilateral                 Caique        14  VP1 + Oil                                                                             0  3    3     3     2     3                                                      1st site                                                                           2nd site                                                                            2nd site                                                                            3rd site                                                                            3rd site                  Sulfur-crested Cockatoo                                                                     9   Antigen Only                                                                          0  0    0     0     0     0                         African Grey Parrot                                                                         21  Antigen Only                                                                          0  0    1     0     2     0                                                           1st site    3rd site                        Caique        1   E3      0  2    *     *     *     *                                                      1st site                                         Sun Conure    4   E3      0  0    0     2     2     0                                                                 2nd site                                                                            3rd site                        Amazon Parrot 18  E3      0  0    1     2     2     0                                                           2nd site                                                                            2nd site                                                                            2nd site                        Ducorp's Cockatoo                                                                           20  E3      0  0    2     2     2     0                                                           1st site                                                                            1st site                                                                            1st site                        African Grey Parrot                                                                         22  E3      0  2    *     *     *     *                                                      1st site                                         Golden Conure 23  E3      0  2    *     *     *     *                                                      1st site                                         Goffin's Cockatoo                                                                           24  E3      0  0    0     2     2     0                                                                 2nd site                                                                            2nd site                        Pionus Parrot 25  E3      0  0    2     0     0     0                                                           2nd site                                    Ringneck Parakeet                                                                           2   Acemannan                                                                             0  0    0     0     0     0                         Blue and Gold Macaw                                                                         5   Acemannan                                                                             0  0    0     0     0     0                         Ducorp's Cockatoo                                                                           6   Acemannan                                                                             0  0    0     0     0     0                         Caique        7   Acemannan                                                                             0  0    0     0     0     0                         Blue-crowned Conure                                                                         13  Acemannan                                                                             0  0    0     0     0     0                         Umbrella Cockatoo                                                                           15  Acemannan                                                                             0  0    0     0     0     0                         African Grey Parrot                                                                         17  Acemannan                                                                             0  1    0     0     0     0                                                      1st site                                         Ducorp's Cockatoo                                                                           3   Equimune                                                                              0  0    0     0     1     2                                                                       3rd site                                                                            3rd site                  Caique        12  Equimune                                                                              0  0    0     0     0     0                         Caique        26  Equimune                                                                              0  0    1     2     2     2                                                           2nd site                                                                            2nd site                                                                            3rd site                                                                            2nd                       __________________________________________________________________________                                                        site                       *Bird died. 0 = no reaction; 1 = slight reaction (hyperemia, skin             discoloration); 2 = mild reaction (small scab formation, thickening of th     skin); 3 = moderate reaction (subcutaneous mass <0.5 cm in diameter or        <0.5 cm area of necrosis); 4 = severe reaction (>0.5 cm subcutaneous mass     or >0.5 cm area of necrosis).                                            

What is claimed is:
 1. A vaccine which is protective against avianpolyomavirus infection in a bird which is classified as being a memberof the Psittaciformes order, comprising:a) an immunogenic amount of aninactivated avian polyomavirus, wherein the immunogenic amount of theinactivated avian polyomavirus corresponds to a titer of at least 10⁴.5TCID₅₀ for the avian polyomavirus before inactivation; b) an adjuvantsuitable for use in a bird which is classified as being a member of thePsittaciformes order; and c) a pharmaceutically acceptable carrier. 2.The vaccine of claim 1, wherein the adjuvant is a long chainpolydispersed β (1,4) linked mannan polymer interspersed withO-acetylated groups.
 3. The vaccine of claim 1, wherein the adjuvant isa deproteinized highly purified cell wall extract derived from anon-pathogenic strain of Mycobacterium species.
 4. The vaccine of claim2, wherein the adjuvant is ACEMANNAN.
 5. The vaccine of claim 1, whereinthe immunogenic amount of the inactivated avian polyomavirus correspondsto a titer of about 10⁵.8 TCID₅₀ for the avian polyomavirus beforeinactivation.
 6. The vaccine of claim 1, wherein the bird is selectedfrom the group consisting of a macaw, an Amazon parrot, a conure, acockatoo, a Pionus Parrot, and an African Grey Parrot.
 7. A method ofpreventing avian polyomavirus infection in a bird which is classified asbeing a member of the Psittaciformes order, comprising administering tothe bird a vaccine comprising:a) an immunogenic amount of an inactivatedavian polyomavirus; b) an adjuvant suitable for use in a bird which isclassified as a member of the Psittaciformes order; and c) apharmaceutically acceptable carrier.
 8. The method of claim 7, whereinthe adjuvant is a long chain polydispersed β (1,4) linked mannan polymerinterspersed with O-acetylated groups.
 9. The method of claim 7, whereinthe adjuvant is a deproteinized highly purified cell wall extractderived from a non-pathogenic strain of Mycobacterium species.
 10. Themethod of claim 8, wherein the adjuvant is ACEMANNAN.
 11. The method ofclaim 7, wherein the immunogenic amount of the inactivated avianpolyomavirus corresponds to a titer of between 10⁴.5 TCID₅₀ and 10⁷TCID₅₀ for the avian polyomavirus before inactivation.
 12. The method ofclaim 7, wherein the immunogenic amount of the inactivated avianpolyomavirus corresponds to a titer of 10⁵.8 TCID₅₀ for the avianpolyomavirus before inactivation.
 13. The method of claim 7, wherein thebird is selected from the group consisting of a macaw, an Amazon parrot,a conure, a cockatoo, a Pionus Parrot, and an African Grey Parrot.
 14. Amethod of preventing avian polyomavirus infection in a bird which isclassified as being a member of the Psittaciformes order, comprisingadministering to the bird a vaccine comprising:a) an immunogenic amountof an inactivated avian polyomavirus which infects a bird from adifferent species of the Psittaciformes order; b) an adjuvant suitablefor use in a bird which is classified as a member of the Psittaciformesorder; and c) a pharmaceutically acceptable carrier.